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Chemistry and Biology of Aroma and Taste

Kaempferol 3-O-(2'''-O-sinapoyl-#-sophoroside) Causes the Undesired Bitter Taste of Canola/Rapeseed Protein Isolates Christoph Hald, Corinna Dawid, Ralf Tressel, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06260 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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Journal of Agricultural and Food Chemistry

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Kaempferol 3-O-(2-O-sinapoyl-β-sophoroside) Causes

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the Undesired Bitter Taste of Canola/Rapeseed Protein

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Isolates

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Christoph Hald†, Corinna Dawid†, Ralf Tressel§ and Thomas Hofmann†,#,‡*

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†Chair

of Food Chemistry and Molecular and Sensory Science, Technical University of Munich, Lise-Meitner-Str. 34, D-85354 Freising, Germany,

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$

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Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg, Germany,

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#Leibniz-Institute

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for Food Systems Biology at the Technical University of Munich,

Lise-Meitner-Str. 34, D-85354 Freising, Germany and

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‡Bavarian

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Center for Biomolecular Mass Spectrometry, Technical University of

Munich, Gregor-Mendel-Straße 4, D-85354 Freising, Germany.

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17 18 19

Running Title: Bitter tastants in Rapeseed Protein

20 21 22

*

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PHONE

+49-8161-712902

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FAX

+49-8161-712949

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E-MAIL

To whom correspondence should be addressed

[email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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By means of activity-guided fractionation using taste dilution analysis (TDA), LC-

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MS/MS, LC-TOF-MS and 1D/2D-NMR spectroscopy, LC-MS/MS quantitation, dose-

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over-threshold considerations, and sensory spiking experiments, kaempferol 3-O-

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(2’’’-O-sinapoyl-β-sophoroside), exhibiting a bitter taste above the low threshold

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concentration of 3.4 µmol/L, was found for the first time as the key molecule

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contributing to the unpleasant bitter taste of rapeseed (canola) protein isolates. This

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finding opens new avenues for a biorefinery approach targeting an off-taste removal.

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Keywords: Rapeseed, Canola, Bitter taste, taste dilution analysis, kaempferol 3-O-

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(2’’’-O-sinapoyl-β-sophoroside)

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INTRODUCTION

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New cultivars of rapeseed, also known as canola, with reduced levels of erucic acid

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and glucosinolates have made rapeseed (Brassica napus), after soybean (351.3 mio

47

tons), become the second most cultivated oil seed crop in the world with a production

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volume of 71.3 mio tons per year, followed by sunflower (47.8), peanuts (43.1) and

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cottonseed (39.1).1,2 Due to its pleasant seed-like and nutty aroma, rapeseed oil is

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today the third most consumed vegetable oil on a global scale.3,4

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With an estimated potential of 1.12 mio tons of crude protein per year, protein-

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rich rapeseed meal generated during oil extraction is also considered an interesting

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domestic protein source exhibiting preferred techno-functionalities and comprising a

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well-balanced amino acid composition of high nutritional value. Although containing

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some anti-nutritive components, such as, e.g. glucosinolates, phenolic compounds,

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and phytates, the high protein value makes rapeseed meal a competitive product in

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the animal feed market.4,5

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As the global food demand will more than double by 2050, protein has been

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identified as a limiting macronutrient in human nutrition and for global food security.6

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Population pressures, ecological considerations and efficiency gains suggest a

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rational evolution from animal to plant protein sources for human nutrition. Despite

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decades of research, several technologies being developed, and products being

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brought to large scale production, there are still no commercially available canola

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protein products, primarily due its intense bitter off-taste limiting palatability in human

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consumption. Although glucosinolates like progoitrin, gluconapin, and glucobrassicin,

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as well as phenolic compounds like sinapine have been reported to exhibit a bitter

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taste,7,8,9 it is still unclear whether these compounds or previously unknown 3 ACS Paragon Plus Environment


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phytometabolites play a key role in the undesired bitter taste of rapeseed protein

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isolates. The knowledge of the key molecules causing the bitter taste of rapeseed

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protein isolates would open new avenues for a targeted bio-refinery approach

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delivering sensorially “clean” protein isolates suitable for rapeseed protein-containing

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food and beverage products with superior taste profiles.

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In recent years, application of a taste-guided fractionation approach enabled

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the identification of the key taste and off-taste compounds in carbohydrate/amino acid

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mixtures10, carots11, black tea infussions12, coffee13, linseed oil14, gouda cheese15,

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asparagus,16 oat,17 and hazelnuts.18 The aim of the present investigation was,

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therefore, to identify the key molecules contributing to the undesired bitter taste of

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rapeseed protein isolates by means of an activity-directed approach, to determine

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their human recognition thresholds, and to evaluate its sensory contribution by means

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of concentration/activity considerations.

81 82 83 84

MATERIALS AND METHODS

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Chemicals. The following compounds were obtained commercially: acetonitrile,

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methanol, and water (J.T. Baker, Deventer, The Netherlands); acetone, ethyl acetate,

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n-pentane (BDH Prolabo, Briare, France); formic acid (Merck, Darmstadt, Germany),

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L-tyrosine

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from bovine milk (Fluka, Steinheim, Germany). Acetonitrile used for HPLC-MS/MS

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analysis was LC-MS grade (Honeywell, Seelze, Germany), acetone, ethyl acetate,

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and n-pentane were distilled before use, all others were HPLC grade. Water for

and deuterated methanol (Sigma-Aldrich, Steinheim, Germany), casein


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chromatography was purified by use of an Advantage A 10 water System (Millipore,

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Molsheim, France). Bottled water (Evian, low mineralization: 405 mg/L) for sensory

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analysis was adjusted to pH 5.9 with formic acid prior to gustatory analysis. Rapeseed

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meal and cruciferin-rich as well as napin-rich protein isolates with a protein content

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of 80-90% were manufactured by Pilot Pflanzenöltechnologie Magdeburg e.V.

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(Magdeburg, Germany) from the rapeseed variety Mentor obtained from

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Norddeutsche Pflanzenzucht Hans-Georg Lembke KG (Holtsee, Germany).

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Sequential Solvent Extraction. An aliquot of the rapeseed protein isolate (300 g)

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was extracted three times with methanol/water (50/50, v/v; 1.5 L) by stirring for 30 min

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at room temperature, followed by centrifugation (5 min, 5000 rpm) and filtration. The

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filtrates were combined, the solvent separated in vacuum at 40 °C and, then,

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lyophilized to give the methanol/water extractables (fraction I). The residue was

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extracted with methanol (1.5 L; fraction II), followed by methanol/acetone (66/33, v/v;

106

1.5 L; fraction III), ethyl acetate (1.5 L; fraction IV), and n-pentane (1.5 L; fraction V).

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The individual solvent fractions I-V were freeze-dried twice to remove trace amounts

108

of solvents and kept at -20°C until used for comparative taste profile analysis (Table

109

1).

110

Solid-Phase-Extraction (SPE) of Fraction I. An aliquot (1 g) of fraction I was

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taken up in water (50 mL) and applied on a Chromabond® C18ec polypropylene

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cartridge (Macherey-Nagel, Düren, Germany) preconditioned with methanol (70 mL),

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followed by water (70 mL). After eluting stepwise with water (75 mL) to give fraction

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I-A, methanol/water (30/70, v/v; 75 mL) to give fraction I-B, methanol/water (50/50,

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v/v; 75 mL) to give fraction I-C, methanol/water (70/30, v/v; 75 mL) to give fraction I-

116

D, and methanol (75 mL) to give fraction I-E. The collected fractions were freed from



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solvent by vacuum evaporation at 40 °C, taken up in water, lyophilized twice and kept

118

at -20 °C until used for sensory analysis (Figure 1).

119

Identification of the Key Bitter Compound in Subfraction I-C. Fraction I-C,

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exhibiting the highest bitter taste activity, was dissolved in acetonitrile/water (20/80,

121

v/v; 5 mg/mL) and, after membrane-filtration, injected onto a 250 x 21 mm, 5 µm,

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Nucleodur C18 Pyramid column (Macherey-Nagel, Düren, Germany). Using a

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flowrate of 20 mL/min and 0.1% formic acid in water (solvent A) and acetonitrile

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(solvent B) chromatography was performed with the following gradient: 0 min 0% B;

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3 min 0% B, 9 min 20% B, 12 min 20% B, 18 min 30 % B, 26 min 30% B, 30 min

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100% B, 33 min 100% B, 38 min 0% B. Monitoring the effluent at 220 nm, the effluent

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was separated into 18 subtractions, namely I-C-1 to I-C-18 (Figure 2). The

128

corresponding subfractions collected from multiple HPLC runs were combined,

129

separated from solvent in vacuum (40 °C), and lyophilized twice prior to sensory

130

analysis using a taste dilution analysis (TDA). LC-MS and NMR analysis (Figure 3)

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revealed kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside) as the key molecule in the

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most bitter tasting subfraction I-C-8. Although this compound was postulated in

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tronchuda cabbage (Brassica oleracea L. Var. costata DC.) and was identified in a

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transgenic low-sinapine oilseed rape seed (Brassica napus L.) its complete 1H NMR

135

data now could be published for the first time.19, 20

136

Kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside), 1 (Figure 3): LC-MS (ESI-): m/z

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815.3 [M-H]-; LC-MS/MS (DP = -35 V, CE= 42 V): m/z 815 [M-H]- (100 %), 623 (21 %),

138

609

139

[M-H-Sinapoyl-H2O-Glc]-, 8 %), 284 [M-H-Sinapoyl-2Glc]-, 37 %), 254 (37 %);

140

LC-MS-TOF: m/z 815.2138 (measured); m/z 815.2040 (calcd. for [C38H39O20]-;

141

1H-NMR

[M-H-Sinapoyl]-,

90 %),

591

[M-H-Sinapoyl-H2O]-,

53 %),

429

(400 MHz; CD3OD): δ 7.92 [“d”,2 H “J” = 8.9 Hz, H-C(2’/6’)], 7.39 [d, 1H, J7’’’’,


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= 16 Hz, H-C(7’’’’)], 6.91 [“d”, 2H, “J” = 8.9 Hz, H-C(3’/5’)], 6.40 [s, 2H,

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8’’’’

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H-C(2’’’’/6’’’’)], 6.19 [d, 1H, ”J” = 16 Hz, H-C(8’’’’)], 6.16, 6.15 [d × 2, 2H, J6,8 = 2.1, J6,8

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= 2.1 Hz, H-C(6/8)], 6.03 [d, 1H, J1’’,2’’ = 8 Hz, H-C(1’’)], 5.28 [d, 1H, J1’’’,2’’’ = 8 Hz,

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H-C(1’’’)], 4.95 [dd, 1H, J2’’’,3’’’ = 9.8 Hz, H-C(2’’’)], 3.95 [dd, 1H, J6’’’A,6’’’B = 12.3, J6’’’A,5’’’

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= 1.9 Hz, H-C(6’’’A)], 3.81–3.76 [m, 3H, H-C(3’’/3’’’/6’’A)], 3.73 (m, 1H, H-C(6’’’B)],

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3.67 [s, 6H, H-C(3’’’’/5’’’’OMe)], 3.60 [dd, 1H, J2’’,3’’ = 9.8 Hz, H-C(2’’)], 3.54–3.49 [m,

148

3H, H-C(4’’’/6’’B/5’’’)], 3.30 [s,1H, H-C(4’’)], 3.27 (ddd, 1H, J5’’,4’’ = 10 Hz, J5’’,6’’B = 5.3

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Hz, J5’’,6’’A = 2.1 Hz, H-C(5’’)]. 13C NMR (125 MHz, CD3OD): δ 177.7 [C(4)], 167.0

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[C(9’’’’)], 164.0 [C(7)], 161.4 [C(5)], 159.9 [C(4’)], 156.6 [C(8a)], 156.0 [C(2)], 147.5

151

[C(3’’’’/5’’’’)], 145.2 [(C(7’’’’)], 137.7 [C(4’’’’)], 133.2 [C(3)], 130.6 [C(2′/6′)], 124.7

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[C(1’’’’)], 121.7 [C(1′)], 114.7 [C(3’/5’)], 114.5 [C(8’’’’)], 104.5, 104.4 [C(4a/2’’’’/6’’’’)],

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98.2 [C(6)], 97.6 [C(1’’’)], 96.3 [C(1’’)], 93.1 [C(8)], 80.4 [C(2’’)], 77.03, 76,56

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[C(5’’/5’’’)], 74.6, 74.36 [C(3’’/3’’’)], 73.7 [C(2’’’)], 70.2, 69.9 [C(4’’/4’’’)], 60.9

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[C(6’’/6’’’)], 54.9 [C(3’’’’/5’’’’OMe)].

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Sensory Analysis. Sensory Panel Training and Sample Pretreatment. The

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sensory panel contained twenty-two panelists (11 females, 11 males, 23-30 years in

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age) who had given informed consent to perform sensory tests and were weekly

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trained with reference taste compounds for at least one year to become familiar with

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the used sensory methodologies and to evaluate different chemosensory

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qualities.12, 21, 22 The sensory analyses were performed at 22-25 °C using nose clips

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to avoid cross-model interactions with odorants.

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Taste Profile Analyses. A portion (1.5 g) of the rapeseed protein was suspended

164

in water (25 mL; pH 5.9) and, after centrifugation, the supernatant presented to the

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trained sensory panel. The panel was asked to evaluate the bitter, astringent and

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sour taste perception on a scale from 0 (not detectable) to 5 (strongly detectable). In



167

addition, aliquots of the fractions I-IV as well as the subfractions I-A to I-E were taken

168

up in bottled water (25 mL, pH 5.9) in “natural” concentrations and, then, evaluated

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by the sensory panelists for bitterness and astringency.

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Taste Dilution Analysis (TDA). The subfractions I-C-1 to I-C-18, isolated from an

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aliquot (200 mg) of fraction I-C, was dissolved in bottled water (20 mL, pH 5.9) and,

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then, sequentially diluted 1:1 (v/v) with bottled water (pH 5.9). The diluted fractions

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were presented to the trained sensory panel in ascending concentrations starting with

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the highest dilution level. The panel was asked to mark where there was a first

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detectable difference between a negative control (bottled water, pH 5.9) and the

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sample, and the taste dilution (TD) factor for bitterness was determined.16 The TD-

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factors for each HPLC-fraction, evaluated in two independent sessions each were

178

averaged.

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Human Taste Recognition Thresholds. To determine the threshold concentration,

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at which the bitter taste quality of the compound was just detectable, a two-alternative

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forced choice test (2-AFC) was performed. Therefore, the purified substance 1 was

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solved in bottled water with increasing levels in concentration. The average threshold

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value of 3.4 µmol/L for 1, represents a range from 1.7 to 6.8 µmol/L, obtained by the

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values between individuals and between the independent sessions which differed by

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not more than plus or minus one dilution step.16

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Comparative Sensory Analysis. To investigate the sensory contribution of the

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bitter compound 1 to the bitter off-taste of a rapeseed protein isolate, a portion (713.4

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nmol) of purified kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside) was spiked to a

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suspension of bovine casein (1.5 g) in bottled water (25 mL; pH 5.9) and, then,

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sensorially compared to the bitter taste intensity of a suspension of rapeseed protein

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isolate (1.5 g) in bottled water (25 mL; pH 5.9) on a 5-point scale (Figure 4).


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Quantitation of Kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside), 1. External

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Calibration Curve and Linear Range. To quantitate the bitter compound 1, a stock

194

solution (54.6 µg/L) was prepared in acetonitrile/water (20/80, v/v) and the exact

195

concentration verified by means of quantitative 1H NMR spectroscopy (qNMR).20 The

196

prepared stock solution was diluted 1:2; 1:5; 1:10; 1:100; 1:200; 1:1000; 1:2000 and

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1:10000 with acetonitrile/water (20/80; v/v) and the dilutions then analyzed by means

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of UHPLC-MS/MS using the characteristic MRM transition Q1/Q3 of m/z 815.1/284.2

199

as the quantifier. By plotting the peak area ratios against the concentrations, an

200

external calibration curve (y = 5.34 X 1005x + 9.52 X 1004, R2 = 0.9987) was received.

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Solvent extraction. Rapeseed protein isolates (1 g) were extracted three times

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with methanol/water (70/30, v/v; 25 mL), each for 10 min, whilst ultrasonication. The

203

combined extracts were filtered, separated from solvent in vacuum at 40 °C, the

204

residue taken up in acetonitrile/water (50/50, v/v; 3 mL) and, after 1:10 dilution with

205

acetonitrile/water (50/50, v/v) and membrane-filtration, analyzed by means of UPLC-

206

MS/MS.

207

High-Performance Liquid Chromatography (HPLC). A HPLC (Jasco, Groß-

208

Umstadt, Germany) consisting of two PU-2087 pumps and a UV-2075 UV-Detector

209

and a Rh 7725 type Rheodyne injection valve (Rheodyne, Bensheim, Germany).

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Chrompass Chromatography Data System, version 1.9 was used for data acquisition.

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Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-

212

MS/MS). To elucidate the structure, mass spectra and ion spectra were acquired on

213

an AB Sciex 5500 Qtrap mass spectrometer (Sciex, Darmstadt, Germany) with direct

214

flow infusion. The acquisition and instrumental control were performed with Analyst

215

1.6.2 software (Applied Biosystems, Darmstadt, Germany). The MS system was

216

operated in full-scan mode (negative, ion spray voltage, -4500 V): curtain gas, 35 V;



217

temperature, 400 °C; gas 1, 45 V; gas 2, 65 V; collision-activated dissociation,

218

medium; DP, -150; EP, -10; CE, - 60 and CXP = -9.

219

To generate quantitative data, the MS System was coupled with a Shimadzu

220

Nexera X2 ultraperfomance liquid chromatography (UPLC) System (Sciex,

221

Darmstadt, Germany) consisting of a DGU-20A 5R degasser, two LC30AD pumps

222

and a SIL30AC autosampler (kept at 15°C) a CTO30A column oven (40°C)

223

equipment with a 100 x 2.1 mm i.d., 1.7 µm, Kinetex C18 100 A (Phenomenex,

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Aschaffenburg, Germany) and was performed with Analyst TF 1.6.2 (AB Sciex,

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Darmstadt, Deutschland). Aliquots (2 µL) of the samples were injected into the

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system running at a flow rate of 0.4 mL/min and using 0.1 % formic acid in water and

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0.1 % formic acid in acetonitrile as solvents A and B, respectively, and the following

228

gradient: start with 0% B, hold 0% for 2 min, increase in 3 min to 30% B, hold 30% B

229

for 5 min, increase in 3 min to 100% B, hold 100% for 3 min, decrease in 2 min to 0%

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B and hold for 2 min isocratically.

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UPLC/Time-of-Flight Mass Spectrometry (UPLC/TOF-MS). High-resolution

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mass spectra were obtained by measuring an aliquot of the analyte in

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acetonitrile/water (20/80, v/v, 1 mL) on a TripleTOF 6600 (AB Sciex, Darmstadt) with

234

a DuoSpray Ion Source coupled with a Nexera X2 UPLC System (Shimadzu, Kyoto)

235

equipment with a 100 x 2.1 mm i.d., 1.7 µm, Kinetex XB-C18 100 A (Phenomenex,

236

Aschaffenburg, Germany) and was performed with Analyst TF 1.7.1 (AB Sciex,

237

Darmstadt, Deutschland).

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Nuclear Magnetic Resonance Spectrometry (NMR). A 400 MHz DRX

239

spectrometer (Bruker, Rheinstetten, Germany) with QNP 1H/14N/13C/31P Z-GRD (300

240

K) was used to record 1D/2D-NMR spectra. Samples were dissolved in methanol-d4

241

(600 µL) and chemical shifts are reported in parts per million (ppm) relative to solvent


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signals in the 1H NMR (3.34 and 4.88 ppm) and the 13C NMR (48.12 ppm) spectrum,

243

respectively. For data processing, the Topspin NMR software vers. 3.2 (Brucker) and

244

MestReNova 11.0.1 (Mestrelab Research, Santiago de Compostela, Spain) were

245

used. Quantitative NMR spectroscopy (q-NMR) was performed as reported earlier

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through calibration of the spectrometer by applying the ERETIC 2 tool using the

247

PULCON methodology.25 For absolute quantitation of the bitter compound 1, the

248

isolated signal at 6.40 ppm [s, 2H, H-C(2’’’’/6’’’’)] was integrated using a defined

249

sample of L-tyrosine and its specific resonance signal at 7.10 ppm (m, 2H) as the

250

external standards.

251 252 253

RESULTS AND DISCUSSION

254 255

Aimed at identifying the key molecule evoking the undesired bitter taste of rapeseed

256

protein isolates, a cruciferin-rich rapeseed protein isolate was first evaluated by

257

means of a taste profile analysis. A trained sensory panel was asked to rate the taste

258

intensities of bitter, astringent and sour on a scale from zero (not detectable) to five

259

(strongly detectable) (Table 1). The protein suspension was rated with a high score

260

for bitterness (1.5), sourness (1.5) and astringency (1.5). To gain a first insight into

261

the polarity of the bitter taste compounds, the protein isolate was extracted with

262

different solvents.

263

Sequential Solvent Extraction of Rapeseed Protein Isolate. The rapeseed

264

protein isolate was extracted sequentially with methanol/water (fraction I), methanol

265

(fraction II), methanol/acetone (fraction III), ethyl acetate (fraction IV), followed by

266

pentane (fraction V). Each fraction was separated from solvent in vacuum, taken up 11 ACS Paragon Plus Environment


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in water in “natural” concentrations and analyzed by means of taste profile analysis

268

(Table 1). While fractions III - V showed only low intensities for bitterness and

269

astringency, fraction I was judged with a high score for bitter taste (2.1) and

270

astringency (1.4), followed by fraction II with some lower bitter taste intensity (1.4).

271

Due to the bitter impact of fraction I, this fraction was further used for identification of

272

the key bitter molecule by means of activity-guided fractionation.

273

Activity-Guided Identification of the Key Bitter Compound in Fraction I. To

274

locate the main bitter compounds, fraction I was separated by means of RP-18 SPE

275

to give five subfractions, namely I-A to I-E, which again were used for sensory

276

analysis. Intense bitter taste (2.8) and astringency (1.7) were detected in fraction I-C,

277

while the other fractions were evaluated with taste intensities lower than 1.0.

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Aimed at identifying the key bitter compound, fraction I-C was further fractionated

279

by means of preparative RP18-HPLC with UV detection (220 nm) to give 18

280

subfractions, namely I-C-1 – I-C-18 (Figure 2), which were freed from solvent, taken

281

up in equal amounts of water, and, then, used for TDA in order of ascending

282

concentrations. By far the highest taste dilution (TD)-factors of 128 and 64 were found

283

for astringency and bitterness in fraction I-C-8, while all the other subfractions showed

284

TD-factors of 16 or lower.

285

The main compound (1) eluting in subfraction I-C-8 was purified by re-

286

chromatography and analyzed by LC-MS/MS, UHPLC-TOF-MS, and 1D/2D-NMR

287

experiments.

288

pseudomolecular ion ([M-H]-), thus indicating a molecular mass of 816.2 and a

289

molecular formula of C38H40O20. MS/MS fragmentation in the negative ESI mode

290

showed the fragment ions m/z 609 [M-H-Sinapoyl]-, 591 [M-H-Sinapoyl-H2O]-, 429

UHPLC-TOF-MS

analysis

revealed


m/z

815.2138

as

the

Page 13 of 29


291

[M-H-Sinapoyl-H2O-Glc]-, and 284 [M-H-Sinapoyl-2Glc/kampferol-2H]-, indicating the

292

presence of a sinapoyl, a kaempferol, as well as two hexose moieties (Figure 3, A).

293

The integrals of the signals in the 1H NMR spectrum of compound 1 displayed a

294

total of 30 protons with signals resonating between 3.23 and 7.92 ppm. With the

295

exception of the singlet signal H-C(3’’’’/5’’’’) resonating at 3.67 ppm, the signals

296

between 3.23 and 6.03 ppm were assigned to the hexose protons. The proton signals

297

observed between 6.14 to 7.92 ppm were assigned to the polyphenol protons of the

298

kaempferol and the sinapoyl moiety. The signals observed at 6.03 [H-C(1’’)] and 5.28

299

ppm [H-C(1’’’)] were assigned as anomeric β-configured glucopyranosyl protons

300

showing a coupling constant of 8 Hz. The doublets at 6.19 and 7.39 ppm with a

301

coupling constant of 16 Hz indicated an (E)-configured sinapinic acid structure.

302

Thirty-two carbon signals were recorded between 54.9 and 177.7 ppm in the 13C NMR

303

spectrum. The 14 quaternary carbon signals at 177.7 [C(4)], 167.0 [C(9’’’’)], 164.0

304

[C(7)], 161.4 [C(5)], 159.9 [C(4’)], 156.6 [C(8a)], 156.0 [C(2)], 147.5 [C(3’’’’/5’’’’)],

305

137.7 [C(4’’’’)], and 133.2 [C(3)] were assigned by means of heteronuclear single-

306

quantum correlation spectroscopy (HSQC). The protons of the two methyl groups at

307

3.67 ppm [H-C(3’’’’/5’’’’OMe)] showed connectivity to the phenyl ring system [147.5

308

ppm C(3’’’’/5’’’’] of the sinapoyl component by means of multiple-bond correlation

309

spectroscopy (HMBC). The carbon atoms show a coupling to the aromatic ring

310

protons at 6.40 ppm [H-C(2’’’’/6’’’’)] and the carbon atoms resonating at 104.4 ppm

311

[C(2’’’’/6’’’’)] showed coupling to proton H-C(7’’’’) of the (E)-configured double bond.

312

Furthermore, the ester carbon atom at 167.0 ppm [C(9’’’’)] showed a coupling to the

313

protons of the (E)-configured double bond [7.39 H-C(7’’’’) and 114.5 H-C(8’’’’)], as

314

well as to H-C(2’’’) of the sugar moiety. The carbon atom C(1’’’) at 97.6 ppm was

315

observed to exhibit coupling to the proton H-C(2’’’) and to the proton H-C(2’’) of the



316

second sugar moiety. These data indicate that the sinapinic acid is esterified with the

317

hydroxyl group at position C(2’’’). The proton H-C(1’’) showed a coupling to the carbon

318

atoms C(2’’’) and to C(3), thus indicating that the sophoroside moiety was (31’’)

319

bound to the kaempferol aglycone (Figure 3, B).

320

Taking all spectroscopic data into consideration, the key bitter compound 1 was

321

identified as kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside) as shown in Figure 3.

322

Although this phytochemical has been reported earlier in tronchuda cabbage

323

(Brassica oleracea L. Var. costata DC.) and transgenic low-sinapine oilseed rape

324

(Brassica napus),17,18 this is, to the best of our knowledge, the first report on the bitter

325

taste impact of this sophoroside.

326

To evaluate the human recognition taste threshold of kaempferol 3-O-(2’’’-O-

327

sinapoyl-β-sophoroside), the purity of compound 1 was confirmed by HPLC-MS and

328

quantitative 1H-NMR spectroscopy to be above 98%. Two alternative-choice test

329

revealed a low human bitter taste threshold of 3.4 µmol/L, which is in the same range

330

as dietary high-potency bitter compounds, such as, e.g. limonin (4.0 µmol/L) in

331

oranges,23 asadanin (13.0 µmol/L) in hazelnuts16, and cis-isocohumulone (7.0

332

µmol/L) in beer24, respectively.

333

Quantitation of Bitter Compound 1 in Rapeseed Protein Isolated and

334

Calculation of Dose-over-Threshold (DoT) Factors. To gain first insights into the

335

concentrations of the bitter compound 1 and to correlate the quantitative data with

336

sensory impact, rapeseed meal and a cruciferin- as well as a napin-rich rapeseed

337

protein isolate were sensorially analyzed in bitter intensity by means of taste profile

338

analysis and extracted with methanol/water and analyzed by UPLC-MS/MS using a

339

solution of the purified kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside) as the

340

external standard (Table 2). The cruciferin-rich protein, exhibiting the strongest bitter


Page 14 of 29

Page 15 of 29


341

off-taste (1.5), also showed the highest levels of compound 1 (475.6 µmol/kg). In

342

comparison, rapeseed meal and the napin-rich protein fraction both showed only low

343

levels of 32.0 and 32.9 µmol/kg of compound 1, being well in line with the low bitter

344

taste intensity judged with a score of 0.8.

345

In order to assess the bitter taste activity of compound 1 in the different protein

346

samples, dose over threshold (DoT)-factors were determined as ratio of the

347

concentration to the taste threshold of the respective tastant.26-30,All three protein

348

isolates exhibited high DoT-factors for compound 1; the most intensely bitter tasting,

349

cruciferin-rich protein isolate showed a DoT-factor of 140.9, while for the less bitter

350

rapeseed meal and napin-rich isolate lower DoT-factors of 9.5 and 9.7, respectively,

351

were determined (Table 2).

352

To confirm the key contribution of kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside)

353

to the undesired bitter taste of rapeseed protein isolates, an aqueous suspension of

354

bovine casein was spiked with compound 1 to match the levels found in an aqueous

355

suspension of the cruciferin-rich rapeseed protein. Sensory analysis of the casein

356

suspension without and with spiked compound 1 as well as the rapeseed protein

357

isolate revealed the same bitter taste intensity (1.5) for the latter two models, while

358

the non-spiked casein solution did not show any significant bitter taste (Figure 4).

359

These data clearly confirm the key contribution of compound 1 to the unpleasant

360

bitter taste of rapeseed protein isolates. The knowledge of kaempferol 3-O-(2’’’-O-

361

sinapoyl-β-sophoroside) as the key bitter molecule will open new avenues for a

362

targeted bio-refinery approach delivering sensorically “clean” protein isolates suitable

363

for rapeseed protein-containing food and beverage products with superior taste

364

profiles.

365



Page 16 of 29

366 367

Funding

368

The project was funded by the Federal Ministry of Education and Research of

369

Germany (BMBF) under the grant number 031B0198D (RaPEQ).

370 371

Notes

372

The authors declare no competing financial interest.

373 374

ACKNOWLEDGMENTS

375

The authors acknowledge the financial support by the Federal Ministry of Education

376

and Research of Germany in the framework of RaPEQ (031B0198D). We are thankful

377

to the whole RaPEQ team, and to Karin Kleigrewe from the Bavarian Center for

378

Biomolecular Mass Spectrometry for measuring the UPLC/Time-of-Flight Mass

379

spectra.

380 381 382 383

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Daun, J. K. Glucosinolate levels in Western Canadian Rapeseed and Canola.

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(in million metric tons). Statista. Accessed 28 March 2018. Available from

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https://www.statista.com/statistics/263937/vegetable-oils-global-consumption/.

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Pastuszeweka, B.; Jablecki, G.; Swiech, E.; Buraczewska, L.; Ochtabinska, A.

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citric acid. Animal Feed Science and Technology. 2000, 86, 117-123

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Breakdown Products. J.Sci.Food Agric. 1983, 34, 73-80

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El-Din Saad El-Beltagi, H.; Mohamed, A.A. Heaney, R.K. Variations in fatty acid

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oil seed rape (Brassica napus L.) cultivars. Grasas Aceites 2010, 111, 55-72

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foods. J. Agric. Food Chem. 2001, 49, 231-238.

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compounds in black tea infusions by combining instrumental analysis and

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bitter metabolites in Gouda cheese. 2008. J. Agric. Food Chem. 2008, 56, 2795-

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Asparagus (Asparagus officinalis L.). J. Agric. Food Chem. 2012, 60, 11877-

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(17) Günther-Jordanland, K.; Dawid, C.; Dietz, M.; Hofmann, T. Key Phytochemicals

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Contributing to the Bitter Off-Taste of Oat (Avena sativa L.). J. Agric. Food

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Chem. 2016, 64, 9639-9652.

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(18) Singldinger, B.; Dunkel, A.; Hofmann, T. The Cyclic Diarylheptanoid Asadanin

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as the Main Contributor to the Bitter Off-Taste in Hazelnuts (Corylus avellana

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L.). J. Agric. Food Chem. 2017, 65, 1677-1683.

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R.M.; Andrade, P.B. Antioxidative properties of tronchuda cabbage (Brassica

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oleracea L. Var. costata DC) external leaves against DPPH, superoxide radical,

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hydroxyl radical and hypochlorous acid. Food Chem., 2006, 98, 416-425

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Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica

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napus). Phytochemistry. 2010, 71, 1076-1084

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steroidal saponins from Asparagus apears (Asparagus officinalis L.), J. Agric.

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Food Chem. 2012, 60, 11889–11900.

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activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao), J.

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Agric. Food Chem. 2005, 53, 5419–5428.

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(23) Glabasnia, A.; Hofmann, T. On the non-enzymatic liberation of limonin and C17-

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epilimonin from limonin-17-β-d-glucopyranoside in orange juice. European Food

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Research and Technology. 2008, 228, 55–63.

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Three TAS2R Bitter Taste Receptors Mediate the Psychophysical Responses



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to Bitter Compounds of Hops (Humulus lupulus L.) and Beer. Chemosensory

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Percept. 2009, 3, 118–132.

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(25) Frank, O.; Kreissl, J. K.; Daschner, A.; Hofmann, T. Accurate determination of

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reference materials and natural isolates by means of quantitative 1H NMR

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spectroscopy. J. Agric. Food Chem. 2014, 62, 2506−2515.

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(26) Hufnagel, J. C.; Hofmann, T. Quantitative reconstruction of the nonvolatile sensometabolome of a red wine, J. Agric. Food Chem. 2008, 56, 9190–9199.

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(27) Scharbert, S.; Hofmann, T. Molecular definition of black tea taste by means of

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quantitative studies, taste reconstitution, and omission experiments, J. Agric.

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Food Chem. 2005, 53, 5377–5384.

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(28) Glabasnia, A.; Hofmann, T. Sensory-directed identification of taste-active

472

ellagitannins in American (Quercus alba L.) and European oak wood (Quercus

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robur L.) and quantitative analysis in bourbon whiskey and oak-matured red

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wines, J. Agric. Food. Chem. 2006, 54, 3380–3390.

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(29) Hillmann, H.; Mattes, J.; Brockhoff, A.; Dunkel, A.; Meyerhof, W.; Hofmann, T.

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Sensomics analysis of taste compounds in balsamic vinegar and discovery of

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5-acetoxymethyl-2-furaldehyde as a novel sweet taste modulator, J. Agric. Food

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Chem. 2012, 60, 9974–9990.

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(30) Sonntag, T.; Kunert, C.; Dunkel, A.; Hofmann, T. Sensory-guided identification

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of N-(1-methyl-4-oxoimidazolidin-2-ylidene)-α-amino acids as contributors to

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the thick-sour and mouth-drying orosensation of stewed beef juice, J. Agric.

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Food Chem. 2010, 58, 6341–6350.

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488 489

Figure Captions

490

Figure 1.

Sensory analysis of SPE fractions I-A to I-E isolated from rapeseed

491

protein isolate. Error bars indicate the 95% confidence interval of the

492

arithmetical mean.

493 494

Figure 2.

RP-HPLC chromatogram (=220 nm; left hand side) of SPE fraction IC and taste dilution analysis (TDA; right hand side).

495 496 497

Figure 3.

(A) MS/MS spectrum, (B) HMBC spectrum (400 MHz, MeOD) and

498

chemical structure of kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside)

499

(1).

500 501

Figure 4.

Bitter taste intensity of a suspension of bovine caseine (1.5 g) in bottled

502

water (25 mL; pH 5.9) before (A) and after spiking with kaempferol 3-

503

O-(2’’’-O-sinapoyl-β-sophoroside) (1; 713.4 nmol) (C) compared to a

504

suspension of cruciferin-rich rapeseed protein isolate (1.5 g; in 25 mL

505

water; pH 5.9) (B) exhibiting a bitter off-taste and containing the same

506

amount of compound 1. Error bars indicate the 95% confidence interval

507

of the arithmetical mean.

508 509 510


Page 22 of 29

Page 23 of 29

511


Table 1. Sensory Evaluation of Fractions Isolated from Rapeseed Protein. Taste intensitya of Fractionb

512 513 514 515

bitterness

astringency

sour

rapeseed proteinc

1.5

1.5

1.5

fraction I

2.1

1.4

1.2

fraction II

1.4

1.0

0.4

fraction III

0.9

0.7

0.4

fraction IV

0.8

0.8

0.5

fraction V

0.3

0.3

0.2

aThe

taste intensity of the given taste descriptors was rated by a trained panel on a scale from 0 (not detectable) to 5 (intensely detectable). bThe panel was asked to rate aqueous solutions of the “natural” concentrations of the fractions I-V. c A cruciferin-rich protein isolate (90% protein) was used for the study.

516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531



Page 24 of 29

532

Table 2. Concentrations, Dose-over-Threshold (DoT) Factors, and Bitter Taste

533

Intensity of Compound 1 in Rapeseed Meal and Protein Isolates, Respectively. Sample

534 535 536 537 538 539

Conc. (µmol/kg) of 1a

DoT factor of 1b

Bitter intensityc

Rapeseed meal

32.0

9.5

0.8

Cruciferin-rich protein isolated

475.6

140.9

1.5

Napin-rich protein isolated

32.9

9.7

0.8

aConcentration

of kaempferol 3-O-(2’’’-O-sinapoyl-β-sophoroside) determined by means of LC-MS/MS (average of triplicates). bDoT factor was calculated as concentration over human taste threshold (3.4 µmol/L). c Bitter taste intensity was received by asking a trained panel to rate different aqueous rapeseed proteins suspensions on a scale from 0 (not detectable) to 5 (intensely detectable). d Rapeseed protein isolates were prepared from rapeseed meal.

540 541 542 543 544 545 546 547 548 549 550 551


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552 553


Hald et al. (Figure 1)

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576


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600



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624


625 626

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